Chemically modified polypeptides

ABSTRACT

The present invention relates to a chemically modified polypeptide in which at least one of hydroxyl groups in the polypeptide molecule is modified with a polyalkylene glycol derivative; a method for producing the modified polypeptide; a method of treatment using the modified polypeptide; use of the modified polypeptide; a pharmaceutical preparation comprising the modified polypeptide; and a composition for treatment comprising the modified polypeptide.

TECHNICAL FIELD

The present invention relates to a chemically modified polypeptide inwhich at least one of the hydroxyl groups in the polypeptide molecule ismodified with a polyalkylene glycol derivative; a method for producingthe modified polypeptide; a method of treating a patient having reducedgranulocytes or thrombocytes using the modified polypeptide; acomposition for the treatment comprising the modified polypeptide; anduse of the modified polypeptide.

BACKGROUND ART

A chemically modified polypeptide in which at least one of amino groups,carboxyl groups, mercapto groups or guanidino groups in the polypeptidemolecule is modified with a polyalkylene glycol derivative (WO95/023165) and modification of free thiol groups of cysteine residues inthe polypeptide molecule are known (EP 0668353). However, when at leastone of such amino groups, carboxyl groups, mercapto groups, guanidinogroups or free thiol groups of cysteine residues in the polypeptidemolecule is modified with a polyalkylene glycol derivative, the activityof the polypeptide may be markedly or completely lost.

For example, the activity of interleukin-15 completely disappears whenits amino group(s) are modified with polyethylene glycol [J. Biol.Chem., 272:2312 (1997)].

Nothing is known about a chemically modified polypeptide in which atleast one of the hydroxyl groups in the polypeptide molecule is modifiedwith a polyalkylene glycol derivative.

Great attention has been directed toward the development of

(1) a method for the analysis of influences of hydroxyl groups upon theactivity of a polypeptide, in a case where the hydroxyl group concernedis in the active site of the polypeptide,

(2) a novel chemical modification method which can avoid a probable casewhere the biological activity of a polypeptide is considerably spoiledwhen the polypeptide is treated by a conventional chemical modificationmethod, and a chemically modified polypeptide obtained by the method,and

(3) a novel method which can improve resistance of polypeptide againstprotease, freezing-thawing and denaturing agents.

DISCLOSURE OF THE INVENTION

The present invention relates to a chemically modified polypeptide inwhich at least one of the hydroxyl groups in the polypeptide molecule ismodified with a polyalkylene glycol derivative; a method for producingthe modifying polypeptide; a method of treating a patient having reducedgranulocytes or thrombocytes using the modifying polypeptide; use of themodifying polypeptide; a pharmaceutical preparation comprising themodifying polypeptide; and a composition for the treatment comprisingthe modifying polypeptide.

With regard to the polypeptide which can be used in the presentinvention, any polypeptide can be used so long as it contains a hydroxylgroup and has a physiological activity or a pharmacological activity.Examples include those having an activity, such as asparaginase,glutaminase, uricase, superoxide dismutase, lactoferin, streptokinase,plasmin, adenosine deaminase, interleukin-1 to 13, interleukin-15,interferon-α, interferon-β, interferon-γ, human granulocytecolony-stimulating factor (hereinafter referred to as “hG-CSF”), and thelike.

Examples of a polypeptide having an hG-CSF activity include apolypeptide comprising the amino acid sequence represented by SEQ IDNO:1, a polypeptide comprising a partial amino acid sequence of thesequence, a polypeptide comprising an amino acid sequence in which someparts of amino acids of the sequence are substituted by different aminoacids [Nature, 319:415 (1986), Japanese Published Unexamined PatentApplication No. 267292/88, Japanese Published Unexamined PatentApplication No. 299/88, WO 87/01132] and the like. Specific examples ofthe polypeptide comprising an amino acid sequence in which some parts ofamino acids of the amino acid sequence represented by SEQ ID NO:1 aresubstituted by different amino acids (hG-CSF derivatives) are shown inTable 1.

TABLE 1 Position from the N-terminal amino acid (hG-CSF of SEQSubstituted amino acid in various hG-CSF derivatives ID NO:1) a) b) c)d) e) f) g) h) i) j) k) l)  1st (Thr) * Val Cys Tyr Arg * Asn Ile Ser *Ala *  3rd (Leu) Glu Ile Ile Ile Thr Thr Glu Thr Thr * Thr *  4th (Gly)Lys Arg Arg Arg Arg Arg Arg Arg Arg Arg Tyr *  5th (Pro) Ser Ser Ser SerSer Ser Ser Ser Ser * Arg * 17th (Cys) Ser Ser Ser Ser Ser Ser Ser SerSer Ser Ser Ser *Unsubstituted amino acid

Examples of the polyalkylene glycol derivative include polyethyleneglycol derivatives, polypropylene glycol derivatives, polyethyleneglycol-polypropylene glycol copolymer derivatives, and the like.

The chemically modified polypeptide of the present invention can beproduced using a chemical modifying agent comprising the abovepolyalkylene glycol derivative, and compounds represented by thefollowing formula (I) can be exemplified as preferred chemical modifyingagents.

The compounds include polyalkylene glycol derivatives represented by

R¹—(M)_(n)—X—R²  (I)

{wherein R¹ represents an alkyl group or an alkanoyl group; M represents

—OCH₂CH₂—, —OCH₂CH₂CH₂—

or

—(OCH₂CH₂)_(r)—(OCH₂CH₂CH₂)_(s)—

(wherein r and s are the same or different, and each represents anoptionally changeable positive integer); n is an optionally changeablepositive integer; X represents a bond, O, NH or S; and R² represents

<wherein R³ represents OH, halogen or

—X^(a)—(M^(a))_(na)—R^(1a)

(wherein X^(a), M^(a), R^(1a) and na each has the same meanings as theabove X, M, R¹ and n, respectively); and Y represents halogen or

—Z—(CH₂)_(p)—(O)_(m)—W

[wherein Z represents O, S or NH; and W represents a carboxyl group or areactive derivative thereof, or

(wherein R⁴ represents an alkyl group, and Hal represents halogen); p isan integer of 0 to 6; and m is 0 or 1]>,

—(CO)_(m)—(CH₂)_(t)—W

(wherein t is an integer of 0 to 6; and m and W have the same meaningsas defined above),

(wherein Hal^(a), pa and R^(4a) each has the same meanings as the aboveHal, p and R⁴, respectively),

(wherein R³ and W have the same meanings as defined above),

(wherein R³, t and W have the same meanings as defined above),

(wherein W has the same meaning as defined above),

(wherein R⁵ represents a residue in which an amino group and a carboxylgroup are removed from an amino acid; and W has the same meaning asdefined above)}.

With regard to the above compound represented by formula (I), examplesof the alkyl group represented by R¹, R⁴ or the like include straight orbranched alkyl groups having 1 to 18 carbon atoms, such as methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, hexyl, isohexyl, heptyl, octyl, isooctyl, decyl,dodecyl, tetradecyl, hexadecyl, octadecyl, and the like; examples of thealkanoyl group represented by R¹ include straight or branched alkanoylgroups having 1 to 18 carbon atoms, such as formyl, acetyl, propionyl,butyryl, valeryl, pivaloyl, pentanoyl, lauroyl, myristoyl, palmitoyl,stearoyl, and the like; examples of the halogen represented by R3, Y,Hal or the like include chlorine, bromine and iodine atoms; examples ofthe reactive derivative of a carboxyl group represented by W or the likeinclude acid halides, such as an acid chloride, an acid bromide, and thelike, active esters, such as a p-nitrophenyl ester, an N-oxysuccinimideester, and the like, and mixed acid anhydrides with monoethyl carbonate,monoisobutyl carbonate and the like; and examples of the amino acidrepresented by R⁵ include glycine, L-alanine, L-valine, L-leucine,L-serine, D-alanine, D-valine, D-leucine, D-serine, β-alanine, and thelike. The positive integer represented by n, r or s is 1 to 20,000,preferably 50 to 5,000 for n, and 1 to 5,000 for r and s.

The polyalkylene glycol derivatives have a molecular weight of 500 to1,000,000, preferably 3,000 to 1,000,000.

A plurality of hydroxyl groups may be present in the polypeptidemolecule, and chemical modification of at least one of these groups maybe sufficient when the polypeptide is chemically modified.

Examples of the hydroxyl group in the polypeptide molecule include ahydroxyl group of a serine or threonine residue, preferably a hydroxylgroup of a serine residue.

The polypeptide can be chemically modified by reacting a chemicalmodifying agent, such as a polyalkylene glycol derivative selected froma group consisting of polyethylene glycol derivatives, polypropyleneglycol derivatives, polyethylene glycol-polypropylene glycol copolymerderivatives and the like, with a polypeptide having a hydroxyl group.

Examples of the method for reacting the hydroxyl group in a polypeptidemolecule with a chemical modifying agent such as polyethylene glycolderivatives or polypropylene glycol derivatives include the methodsdescribed in Japanese Published Unexamined Patent Application No.316400/89, Biotech. Lett., 14:559-564 (1992), BIO/TECHNOLOGY, 8:343-346(1990), and the like, and modified methods thereof. That is, specificexamples of the methods which can be used include methods wherein apolyethylene glycol derivative or a polypropylene glycol derivative isadded to an aqueous solution of a protein which has been adjusted to apH of 6 to 10 in an amount of 1 to 200 moles per protein, and is allowedto react at a temperature of 0 to 37° C. for 1 hour to 3 days.

Examples of the method for reacting the hydroxyl group in a polypeptidemolecule with a polyethylene glycol-polypropylene glycol copolymerderivative include the methods described in Japanese PublishedUnexamined Patent Application No. 59629/84, Japanese PublishedUnexamined Patent Application No. 176586/85, WO 89/06546, EP 0539167A2,and the like, and modified methods thereof. That is, specific examplesof the methods which can be used include methods wherein a chemicalmodifying agent selected from a group consisting of polyethyleneglycol-polypropylene glycol copolymer derivatives is added to an aqueoussolution of a protein which has been adjusted to a pH of 6 to 10 in anamount of 1 to 200 moles per protein, and is allowed to react at atemperature of 0 to 37° C. for 1 hour to 3 days.

Modification of at least one hydroxyl group in the polypeptide moleculewith a polyalkylene glycol derivative by the above method can provide

(1) a method for the analysis of influences of the hydroxyl group uponthe activity of a polypeptide, in a case where the hydroxyl groupconcerned is in the active site of the polypeptide,

(2) a novel chemical modification method which can avoid diminishing thebiological activity of a polypeptide when the polypeptide is treated bya conventional chemical modification method, and a chemically modifiedpolypeptide obtained by the method, and

(3) a novel method which can improve a polypeptide's resistance toprotease, freezing-thawing or denaturing agents, and a chemicallymodified polypeptide having improved resistance against protease,freezing-thawing or denaturing agents.

The chemically modified polypeptide of the present inventionspecifically described is an example in which a polypeptide having anhG-CSF activity is used as a polypeptide.

A chemically modified polypeptide in which at least one hydroxyl groupin hG-CSF or a hG-CSF derivative is chemically modified with a chemicalmodifying agent represented by the following formula (Ia) or (Ib) can beexemplified as the chemically modified polypeptide of the presentinvention.

Chemical modifying agent (Ia):

R¹—(OCH₂CH₂)_(n)—X—R^(2a)  (Ia)

{wherein R¹, n and X have the same meanings as defined above; and R^(2a)represents

[wherein R represents OH, halogen or

—X^(b)—(CH₂CH₂O)_(nb)—R^(1b)

(wherein X^(b), R^(1b) and nb each has the same meanings as the above X,R¹ and n, respectively)]}.

Chemical modifying agent (Ib):

(wherein R¹, M, R³, Z, n and p have the same meanings as defined above;and W^(a) represents a carboxyl group or a reactive derivative thereof).

At least one molecule of polyethylene glycol derivatives, polypropyleneglycol derivatives or polyethylene glycol-polypropylene glycol copolymerderivatives is bonded to a chemically modified hG-CSF or a chemicallymodified hG-CSF derivative. Thus, the chemically modified hG-CSF or thechemically modified hG-CSF derivative can be used as a mixture or byseparating a compound to which one or more molecules are attached.

Separation of the chemically modified hG-CSF or the chemically modifiedhG-CSF derivative can be carried out using various types ofchromatography, such as ion exchange chromatography, gel filtrationchromatography, reverse phase chromatography, hydrophobicchromatography, and the like, and methods, such as ammonium sulfatefractionation and the like, which are generally used for the separationof long chain polypeptides and the like.

The degree of chemical modification can be confirmed by monitoringchanges in the mobility of the chemically modified hG-CSF using a sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method.

The polypeptide content according to the present invention can bemeasured by the following assay methods.

Assay method 1:

The polypeptide content is measured by the Lowry method [Lowry O. H. etal., J. Biol. Chem., 193:265 (1951)].

Assay method 2:

The polypeptide content is calculated by carrying out SDS-PAGE inaccordance with the method of Laemmli [U. K. Laemmli, Nature, 227:680(1970)], staining the polypeptide separated on the gel with CoomassieBrilliant Blue and then measuring it by a chromatoscanner (CS-930,Shimadzu Corp.).

The chemically modified polypeptide of the present invention can be usedas such or in various dosage formulations.

The pharmaceutical preparations of the present invention can be producedby mixing an effective amount of a chemically modified polypeptide asthe active ingredient uniformly with a pharmaceutically acceptablecarrier. It is preferred that these pharmaceutical preparations are in aunit dosage form suitable for injection administration.

Injections can be prepared as solutions using a chemically modifiedpolypeptide and a carrier comprising distilled water, a salt solution, aglucose solution or a mixture of a salt solution and a glucose solution.They are also prepared as solutions, suspensions or dispersions in theconventional way using appropriate auxiliaries. They can be alsoprepared as freeze-dried preparations by freeze-drying the solutions.Although freeze-drying conditions are not particularly limited, afreeze-dried product is generally obtained by freezing at −50° C. orless for 1 to 5 hours, drying at a shelf temperature of −20° C. to 0° C.for 24 to 48 hours under a vacuum degree of 50 to 150 mTorr and thendrying at a shelf temperature of 10° C. to 30° C. for 16 to 24 hoursunder a vacuum degree of 50 to 100 mTorr.

Also, the chemically modified polypeptide preparations can containvarious generally used pharmaceutical carriers, fillers, diluents,stabilizers, adsorption preventing agents and the like.

In the case of a chemically modified polypeptide of the presentinvention, for example a neutrophil and thrombocyte growth enhancingpreparation containing a chemically modified hG-CSF or a chemicallymodified hG-CSF derivative, its dose and its administration schedule aredecided depending on its mode of administration, age and body weight ofeach patient, the disease to be treated and morbid state of eachpatient; however, a pharmaceutical preparation containing a chemicallymodified hG-CSF or a chemically modified hG-CSF derivative in an amountof 15 μg to 1.5 mg, preferably 25 to 500 μg, per adult is usuallyadministered 1 to 7 times per week.

Examples of the administration method of the chemically modifiedpolypeptide preparation of the present invention include intravenousinjection, subcutaneous injection, and the like, as well asadministration as suppositories or nasal drops.

Next, pharmacological activities of the chemically modified polypeptideof the present invention are described by Test Examples.

Test Example 1

Growth promoting activity of chemically modified hG-CSF and chemicallymodified hG-CSF derivatives upon mouse leukemia cell NFS60:

The activity of the G-CSF derivative, chemically modified hG-CSFderivative and chemically modified G-CSF obtained in Reference Example 1and Examples 5, 14, 17 and 20, which will be described later, to enhancegrowth of mouse leukemia cell NFS60 [K. Holmes et al., Proc. Natl. Acad.Sci. USA, 82:6687 (1985)] was measured in accordance with the method ofAsano et al. [Yakuri to Chiryo, 19:2767 (1991)]. Each compound inrespective concentrations shown in Tables 2-1 and 2-2 was allowed to actupon the cells, with the results also shown in Tables 2-1 and 2-2.

TABLE 2-1 Conc. NFS60 cell growth Compound (ng/ml) promoting activity(%)*¹ hG-CSF derivative of Ref. Ex. 1 12.5 100 Mono-substituted 1 of Ex.5 12.5 100 Mono-substituted 2 of Ex. 5 12.5 100 Di-substituted of Ex. 512.5 100 Tri-substituted 1 of Ex. 5 12.5 72 Tri-substituted 2 of Ex. 512.5 93 hG-CSF 5 100 Mono-substituted 1 of Ex. 14 5 86 Mono-substituted2 of Ex. 14 5 81 Di-substituted of Ex. 14 5 81 *¹Shown by a relativevalue (%) based on the activity (= 100) of the hG-CSF derivative (12.5ng/ml) obtained in Reference Example 1 or of hG-CSF (5 ng/ml).

TABLE 2-2 Conc. NFS60 cell growth Compound (ng/ml) promoting activity(%)*¹ hG-CSF derivative of Ref. Ex. 1 25 100 Tri-substituted 1 of Ex. 1725 92 Tri-substituted 2 of Ex. 17 25 80 Di-substituted of Ex. 17 25 99Tri-substituted 1 of Ex. 20 25 65 Tri-substituted 2 of Ex. 20 25 73Di-substituted of Ex. 20 25 98 *¹Shown by a relative value (%) based onthe activity (= 100) of the hG-CSF derivative (25 ng/ml) obtained inReference Example 1.

Test Example 2

Effect to promote recovery of thrombopenia in total body radiation mice:

Four animals per group of male BALB/c mice (6 weeks of age) wereradiated to the total bodies (hereinafter referred to as “Rx”) with 3 Gyper mouse by a 137 Cs radiation source (RI-433, manufactured by Toshiba)and then reared in a clean rack of a specified pathogen-free (SPF)rearing environment facility. They were freely provided with drinkingwater and feed. As a non-treated control group, mice with no radiationwere reared in a manner similar thereto.

Each of chemically modified hG-CSF derivatives obtained in Example 4which will be described later was dissolved in physiological saline, andthe chemically modified hG-CSF derivative solution was subcutaneouslyadministered to mice once on the next day of Rx in a dose of 5 μg/0.2 mlper animal.

Blood samples were periodically collected from the murine vein ofeyeground to measure the number of platelets by an automatic blood cellcounter (CC-180A, manufactured by Toa Iyo Denshi) The results are shownin Table 3.

In the mice treated with total body radiation of 3 Gy, a considerabledecrease in the number of platelets was observed, and the number ofplatelets became the lowest on 8 to 9 days after Rx and then graduallyincreased but did not recover to the level before the radiationtreatment. On the other hand, decrease in the number of platelets wasinhibited in the mice to which the chemically modified hG-CSF derivativewas administered, and the number of platelets increased markedly on andafter the 8th or 9th day and completely recovered on the 11th or 12thday to the same level before the radiation treatment. The similar effectwas observed in a group in which the administration was carried out onthe next day and 5th day after Rx.

TABLE 3 Chemically Average number of platelets (%)*¹ modified hG-CSFDays after derivative radiation 0 6 8 10 11 12 Not administered 100 58.926.6 35.1 38.3 45.1 Di-substituted 100 55.1 30.5 62.7 98.5 103.6 of Ex.4 *¹Shown by a relative value (%) based on the average number ofplatelets (= 100) in the total body radiation-untreated control group.

Test Example 3

Leukocyte increasing action in mice:

Using SPF/VAF mice (BALB/cAnNCrj line, males, 8 weeks of age, 4 animalsper group) which were preliminarily reared after purchase from CharlesRiver Japan, leukocyte increasing activity in normal mice was confirmed.

Di-substituted obtained in Example 5 (0.34 mg/ml) was diluted to 100 or10 μg/ml using physiological saline, and subcutaneously administeredonce at a rate of 10 μl/g (mouse weight). Thus, the dose was 1 or 0.1mg/kg. As a control group, physiological saline was subcutaneouslyadministered once. Blood samples were collected before theadministration and periodically from the next day after theadministration, and the number of peripheral blood cells was measuredusing an automatic blood cell counter (Sysmex F800).

As the results, the number of leukocytes in both administered groupsincreased to 2.4 to 2.6 times higher level than that of the controlgroup 2 days after the administration. Thereafter, this drug effectattenuated in the 0.1 mg/kg administration group and returned to asimilar level of the control group 4 days after the administration, butthe number of leukocytes in the 1 mg/kg administration group continuedto increase even after 2 days after administration and reached a level3.5 times higher than that in the control group 4 days afteradministration. Thereafter, the number returned to a level similar tothe control group on the 7th day after the administration.

Examples and Reference Examples are shown below.

BEST MODE FOR CARRYING OUT THE INVENTION Example 1

Preparation of polyethylene glycol-modified hG-CSF derivative reactionsolution:

The hG-CSF derivative prepared in Reference Example 1 was adjusted to4.6 mg/ml using a phosphate buffer (pH 7.5), and 12 ml of the thusadjusted solution was mixed with 205.5 mg of N-hydroxysuccinimide esterof monomethoxypolyethylene glycol propionate (M-SPA-20,000, manufacturedby Shearwater Polymer) and the mixture was stirred at 4° C. for a wholeday and night to prepare a polyethylene glycol-modified hG-CSFderivative reaction solution (1).

Example 2

Preparation of polyethylene glycol-modified hG-CSF derivative reactionsolution:

The hG-CSF derivative prepared in Reference Example 1 was adjusted to4.6 mg/ml using a phosphate buffer (pH 7.5), and 12 ml of the thusadjusted solution was mixed with 469.8 mg of N-hydroxysuccinimide esterof carboxymethyl-monomethoxypolyethylene glycol (M-SCM-20,000,manufactured by Shearwater Polymer) and the mixture was stirred at 4° C.for a whole day and night to prepare a polyethylene glycol-modifiedhG-CSF derivative reaction solution (2).

Example 3

Preparation of polyethylene glycol-modified hG-CSF derivative reactionsolution:

The hG-CSF derivative prepared in Reference Example 1 was adjusted to4.6 mg/ml using a phosphate buffer (pH 7.5), and 6.6 ml of the thusadjusted solution was mixed with 96.9 mg of N-hydroxysuccinimide esterof monomethoxypolyethylene glycol propionate (M-SSPA-20,000,manufactured by Shearwater Polymer) and the mixture was stirred at 4° C.for a whole day and night to prepare a polyethylene glycol-modifiedhG-CSF derivative reaction solution (3).

Example 4

Isolation of polyethylene glycol-modified component:

The polyethylene glycol-modified hG-CSF derivative reaction solution (2)prepared in Example 2 (protein content, 55 mg) was diluted 5 times witha 20 mM acetate buffer (pH 4.5) containing 150 mM of sodium chloride andpassed through a column packed with Sephacryl S-400 (manufactured byPharmacia) which had been equilibrated in advance with the same buffer,and the eluates were fractionated. By this operation, a fraction mainlycontaining a component modified with two molecules of polyethyleneglycol (Di-substituted) and a crude fraction mainly containing acomponent modified with one molecule of polyethylene glycol(Mono-substituted) were obtained.

Each of these fractions was passed through a column packed with TSK gelG4000SW_(XL) (7.8 mm I.D.×300 mm, manufactured by Tosoh Corp.) to obtaina fraction containing Di-substituted or Mono-substituted. Thereafter,each of these fractions was purified using SP-5PW (21.5 mm I.D.×150 mm,manufactured by Tosoh Corp.) under the following conditions to obtainone species of the main component of Di-substituted (0.39 mg/ml×3.0 ml)and one species of the main component of Mono-substituted (0.55mg/ml×3.0 ml).

SP-5PW Purification Conditions:

Column: SP-5PW (21.5 mm I.D.×150 mm) (manufactured by Tosoh Corp.)

Detection: 280 nm

Solution A: 20 mM acetate buffer (pH 4.5)

Solution B: 20 mM acetate buffer (pH 4.5) containing 0.5 M sodiumchloride

Elution: linear density gradient elution from solution A to solution B

Example 5

Isolation of polyethylene glycol-modified component:

The polyethylene glycol-modified hG-CSF derivative reaction solution (1)prepared in Example 1 (protein content, 55 mg) was diluted 5 times witha 20 mM acetate buffer (pH 4.5) containing 150 mM of sodium chloride andpassed through a column packed with Sephacryl S-400 (manufactured byPharmacia) which had been equilibrated in advance with the same buffer,and the eluates were fractionated. By this operation, a crude fractioncontaining a component modified with three molecules of polyethyleneglycol (Tri-substituted), a crude fraction containing a componentmodified with two molecules of polyethylene glycol (Di-substituted) anda crude fraction containing a component modified with one molecules ofpolyethylene glycol (Mono-substituted) were obtained.

Each of these fractions was passed through a column packed with TSK gelG4000SW_(XL) (7.8 mm I.D.×300 mm, manufactured by Tosoh Corp.) to obtaina fraction containing Tri-substituted, Di-substituted orMono-substituted. Thereafter, each of these fractions was purified usinga cation exchange column, SP-5PW (manufactured by Tosoh Corp.), underthe same conditions described in Example 4 to obtain two species of themain component of Tri-substituted (Tri-substituted 1:1.4 mg/ml×0.5 ml,Tri-substituted 2:1.0 mg/ml×0.8 ml), one species of the main componentof Di-substituted (0.34 mg/ml×3.0 ml) and two species of the maincomponents of Mono-substituted (Mono-substituted 1:0.48 mg/ml×0.4 ml,Mono-substituted 2:1.58 mg/ml×0.5 ml).

As will be described in later examples, the hydroxyl group of the 66position Ser counting from the N-terminus of Mono-substituted 1 and theN-terminal Met of Mono-substituted 2 were modified with one molecule ofpolyethylene glycol.

Example 6

Isolation of polyethylene glycol-modified component:

The polyethylene glycol-modified hG-CSF derivative reaction solution (3)prepared in Example 3 (protein content, 30 mg) was diluted 5 times witha 20 mM acetate buffer (pH 4.5) containing 150 mM of sodium chloride andpassed through a column packed with Sephacryl S-400 (manufactured byPharmacia) which had been equilibrated in advance with the same buffer,and the eluates were fractionated. By this operation, a crude fractioncontaining a component modified with two molecules of polyethyleneglycol (Di-substituted) was obtained.

This fraction was passed through a column packed with TSK gelG4000SW_(XL) (7.8 mm I.D.×300 mm, manufactured by Tosoh Corp.) to obtaina fraction containing Di-substituted.

The thus obtained fraction was purified using a cation exchange column,SP-5PW (manufactured by Tosoh Corp.) under the same conditions describedin Example 4 to obtain one species of the main component ofDi-substituted (0.43 mg/ml×3.0 ml).

Example 7

Peptide mapping of hG-CSF derivative:

The hG-CSF derivative prepared in Reference Example 1 was adjusted to 2mg/ml using a phosphate buffer (pH 7.5), a 1.0 ml portion of the thusadjusted solution was treated with V8 protease (manufactured bySeikagaku Kogyo) under the conditions described in “High PerformanceLiquid Chromatography of Protein and Peptide (II), Kagaku Zokan 117(153-160 (1990); published by Kagaku Dojin)” and then a 50 μl portion ofthe thus treated solution was injected into an HPLC to carry out HPLCanalysis under the following conditions.

Conditions for HPLC Analysis:

Column: PROTEIN & PEPTIDE C18 (4.6 mm I.D.×250 mm, VYDAC)

Solution A: 0.1% trifluoroacetic acid

Solution B: 90% acetonitrile containing 0.1% trifluoroacetic acid

Elution: linear density gradient elution from solution A to solution B

Detection: 215 nm

Flow rate: 0.5 ml/min

Nine peaks were found on the analytical pattern of the hG-CSF derivativedigested with V8 protease. When each of the peaks was separated and itsmolecular weight analysis was carried out, these peaks were assigned asshown in Table 4.

TABLE 4 Peptide mapping of hG-CSF derivative by V8 protease Molecularweight analyzed Peak (calculated) No. Peptide fragment *(Na addition) 194-98 524.5* (502) 2 20-33 1512.8 (1514) 3 34-46 1648.8 (1649) 4 163-1741438.9 (1440) 5 −1-19 2241.7 (2241) 6 124-162 4028.0 (4029) 7 105-1232263.8* (2240) 8 47-93 5060.5 (5060) 9  99-123 2836.9 (2836)

Example 8

Estimation of polyethylene glycol binding position by peptide mapping:

Each of Mono-substituted 1, Mono-substituted 2, Di-substituted,Tri-substituted 1 and Tri-substituted 2 obtained in Example 5 wasdigested with a protease by a procedure similar to the hG-CSF derivativepeptide mapping described in Example 7.

As the results, disappearance or considerable reduction of specifiedpeaks was found in Mono-substituted 1, Di-substituted, Tri-substituted 1and Tri-substituted 2 obtained in Example 5 (Table 5).

TABLE 5 Peptide mapping of purified polyethylene glycol- modified hG-CSFderivative component (comparison with unmodified hG-CSF derivative)Disappeared or markedly Component reduced peak No. Mono-substituted 1 ofEx. 5 8 Mono-substituted 2 of Ex. 5 5 Di-substituted of Ex. 5 5, 8Tri-substituted 1 of Ex. 5 3, 5, 8 Tri-substituted 2 of Ex. 5 3, 5, 8

It is believed that the HPLC peaks of the polyethylene glycol-linkedpeptide fragments will change when compared with the analytical patternof the hG-CSF derivative of Example 7. Based on the results of Table 4,it was estimated that the binding position of polyethylene glycol was atleast a peptide residue (including an N-terminal amino group)corresponding to a fragment of −1 to 19 amino acid residues (peak 5) ofthe hG-CSF derivative in the case of Mono-substituted 2, Di-substituted,Tri-substituted 1 and Tri-substituted 2 shown in Example 5.

With regard to Mono-substituted 2, it was found that the N-terminal Metresidue of Mono-substituted 2 was modified with one molecule ofpolyethylene glycol, because its N-terminal sequence was not observedwhen the sequence was measured using a protein sequencer PPSQ-10manufactured by Shimadzu Corp.

With regard to Tri-substituted 1 and Tri-substituted 2, it was estimatedthat polyethylene glycol was also bound to at least a peptide residue(including an amino group of Lys) corresponding to a fragment of 34 to46 amino acid residues (peak 3) of the hG-CSF derivative.

In addition, since the peak 8 disappeared or markedly reduced in each ofMono-substituted 1, Di-substituted, Tri-substituted 1 andTri-substituted 2, it was estimated that polyethylene glycol was alsobound to a peptide residue (excluding free amino groups of lysine andthe like) corresponding to a fragment of 47 to 93 amino acid residues ofeach of these components.

Example 9

Estimation of polyethylene glycol binding position by peptide mapping:

The fraction of Di-substituted obtained in Example 6 was digested with aprotease by a procedure similar to the hG-CSF derivative peptide mappingdescribed in Example 7.

When compared with the analytical pattern of the hG-CSF derivative ofExample 7, disappearance or considerable reduction of the specifiedpeaks shown in Table 6 was found in Di-substituted obtained in Example6.

TABLE 6 Peptide mapping of purified polyethylene glycol- modified hG-CSFderivative component (comparison with unmodified hG-CSF derivative)Disappeared or markedly Component reduced peak No. Di-substituted of Ex.5 5, 8

It is predicted that the HPLC peaks of the polyethylene glycol-linkedpeptide fragments will change when compared with the analytical patternof the hG-CSF derivative of Example 7. Based on the results of Table 5,similar to the case of Example 8, it was estimated that bindingpositions of polyethylene glycol were a peptide residue (including anN-terminal amino group) corresponding to a fragment of −1 to 19 aminoacid residues and a peptide residue (excluding free amino groups oflysine and the like) corresponding to a fragment of 47 to 93 amino acidresidues of the hG-CSF derivative in the case of Di-substituted shown inExample 6.

Example 10

Estimation of polyethylene glycol binding position by peptide mapping:

The binding position of polyethylene glycol on Di-substituted obtainedby a procedure similar to that of Example 4 was examined by peptidemapping in a manner similar to those in Examples 7 and 8. It wasestimated that polyethylene glycol was also bound to a peptide residue(including an N-terminal amino group) corresponding to a fragment of −1to 19 amino acid residues and a peptide residue (excluding free aminogroups of lysine and the like) corresponding to a fragment of 47 to 93amino acid residues of the hG-CSF derivative in the case ofDi-substituted shown in Example 4.

Example 11

Purification of polyethylene glycol-modified peptide:

A fragment of polyethylene glycol-bound peptide was isolated fromDi-substituted component by the following procedure in order to examinepolyethylene glycol binding amino acid residues in the peptide fragmentcorresponding to the 47 to 93 position amino acid residues containing nofree amino groups, of the hG-CSF derivative or hG-CSF, which was thepolyethylene glycol binding position confirmed in Di-substituted ofExamples 4, 5 and 6, Tri-substituted 1 and Tri-substituted 2 of Example5, Mono-substituted 1 of Example 5, Mono-substituted 1 andDi-substituted of Example 14, Di-substituted, Tri-substituted 1 andTri-substituted 2 of Example 17 or Di-substituted, Tri-substituted 1 andTri-substituted 2 of Example 20.

That is, 30 ml of a fraction containing Di-substituted as the maincomponent (protein concentration, 2.2 mg/ml) was obtained by a cationexchange chromatography column, SP-5PW, in a manner similar to that inExample 5 from a polyethylene glycol-modified hG-CSF derivative reactionsolution prepared in a manner similar to that in Example 1. Next, thiswas digested with thermolysin (manufactured by Sigma). Using a gelfiltration column (Sephacryl S-300, manufactured by Pharmacia), theenzyme reaction solution was fractionated to obtain the desiredpolyethylene glycol-bound peptide fraction (about 30 mg).

The result of mass spectrometry (MALDI-TOF MS) of the peptide fractionwas 22155.89 (M+H), and the result of amino acid analysis showed Ser 2.7(3), Pro 1.0 (1), Leu 1.0 (1) and Cys 0.9 (1).

Based on the results of mass spectrometry and amino acid analysis, itwas estimated that the sample isolated by the above procedure was afragment in which polyethylene glycol was bound to a peptide of position61 residue to the 66 position residue [LeuSerSerCysProSer(leucyl-seryl-seryl-S-amidomethylcysteinyl-prolyl-serine) residue] ofthe hG-CSF derivative.

Example 12

Determination of polyethylene glycol binding position by ¹H-NMR:

A peptide, LeuSerSerCysProSer(leucyl-seryl-seryl-cysteinyl-prolyl-serine) corresponding to the 61 to66 position amino acid residues of the hG-CSF derivative was synthesizedby a peptide solid phase synthesis method (PSSM 8, Shimadzu Corp.) andamidomethylated under the conditions shown in “High Performance LiquidChromatography of Protein and Peptide (II), Kagaku Zokan 117 (153-160(1990); published by Kagaku Dojin)” and then the thus amidomethylatedpeptide (leucyl-seryl-seryl-S-amidomethylcysteinyl-prolyl-serine) waspurified by reverse phase HPLC. A 0.6 mg portion of the amidomethylatedpeptide was dissolved in deuterium-substituted dimethyl sulfoxide(d₆-DMSO) to carry out ¹H-NMR analysis (500 MHz). In a similar manner,¹H-NMR analysis was carried out using 20 mg of the polyethyleneglycol-bound peptide obtained in Example 11. Chemical shift of protons(other than those originating from polyethylene glycol) observed by eachanalysis is shown in Tables 7 and 8. From the results, all of theprotons originating from the γOH groups of three Ser residues wereconfirmed in the amidomethylated peptide obtained by synthesis. On theother hand, some phenomena were observed in the polyethyleneglycol-bound peptide obtained in Example 11, in addition to the signalof protons originating from polyethylene glycol, that the protonoriginating from γOH group of the 66 position Ser residue of the hG-CSFderivative was not observed, that β proton of the same Ser residue wasshifted to about 0.6 ppm-lower magnetic field and that amido proton ofthe same Ser residue gave a broad signal in comparison with other amidoprotons. On the basis of the above results, it was confirmed that thebinding position of polyethylene glycol was the 66 position Ser residueof the hG-CSF derivative.

TABLE 7 Chemical shift (ppm) of protons in NMR analysis ofamidomethylated peptide synthesized Residue NH CαH CβH Others Leu 618.06 3.86 1.52, 1.57 γH1.67, δCH₃0.92 Ser 62 8.66 4.47 3.57, 3.64 γOH5.13 8.66 4.49 3.63 γOH5.04 Ser 63 8.03 4.32 3.58 γOH4.85 8.06 4.323.57 γOH4.80 Cys 64 8.19 4.68 2.63, 2.96 CH₂3.12, NH₂7.05, 7.42 7.974.60 2.63, 2.90 CH ₂ 3.12, NH ₂ 7.10, 7.45 Pro 65 4.42 1.91, 2.02γH1.86, δH3.61 4.80 1.98, 2.20 γ H1.83, δH3.45, 3.40 Ser 66 7.98 4.233.60, 3.70 γOH4.94 8.40 4.24 3.80, 3.72 γOH4.90 The italic typerepresents Cis-Pro.

TABLE 8 Chemical shift (ppm, excluding the polyethylene glycol moiety)of protons in NMR analysis of polyethylene glycol-modified peptideisolated from polyethylene glycol-modified hG-CSF derivative Residue NHCαH CβH Others Leu 61 3.66 1.44, 1.56 γH1.70, δCH₃0.90 3.73 1.47, 1.60γH1.67, δCH ₃ 0.90 Ser 62 8.51 4.43 3.57, 3.64 γOH5.17 8.90 4.37 3.64Ser 63 8.03 4.30 3.60 γOH4.85 8.06 4.21 3.58, 3.63 Cys 64 8.18 4.682.68, 3.03 CH₂3.12, NH₂7.02, 7.43 8.08 4.62 2.67, 2.90 CH ₂3.12,NH₂7.02, 7.62 Pro 65 4.33 1.87, 2.02 γH1.86, δH3.67 4.60 2.12, 2.04γH1.72, 1.83, δH3.40, 3.52 Ser 66 7.88 4.28 4.17, 4.32 7.66 4.21 4.16,4.46 The italic type represents Cis-Pro.

Example 13

Preparation of polyethylene glycol-modified hG-CSF reaction solution:

A 4.0 ml portion of a 4.0 mg/ml solution of hG-CSF prepared using aphosphate buffer (pH 7.5) was mixed with 83 mg of N-hydroxysuccinimideester of monomethoxypolyethylene glycol propionate (M-SPA-20,000,manufactured by Shearwater Polymer) and the mixture was stirred at 4° C.for a whole day and night.

Example 14

Isolation of polyethylene glycol-modified component:

The polyethylene glycol-modified reaction solution prepared in Example13 (protein content, 16 mg) was diluted 5 times with a 20 mM acetatebuffer (pH 4.5) containing 150 mM of sodium chloride and fractionatedusing a column packed with Sephacryl S-400 (manufactured by Pharmacia)which had been equilibrated in advance with the same buffer. By thisoperation, a fraction mainly containing a component modified with twomolecules of polyethylene glycol (Di-substituted) and a crude fractionmainly containing a component modified with one molecules ofpolyethylene glycol (Mono-substituted) were obtained. Each of thesefractions was applied to a column packed with TSK gel G4000SW_(XL) (7.8mm I.D.×300 mm, manufactured by Tosoh Corp.) to obtain a fractioncontaining Di-substituted or Mono-substituted. Thereafter, each of thesefractions was purified using SP-5PW (21.5 mm I.D.×150 mm, manufacturedby Tosoh Corp.) to obtain one species of the main component ofDi-substituted (0.61 mg/ml×0.6 ml) and two species of the main componentof Mono-substituted (Mono-substituted 1:0.46 mg/ml×1.0 ml,Mono-substituted 2:0.61 mg/ml×0.7 ml).

Example 15

Estimation of polyethylene glycol binding position by peptide mapping:

Each of Mono-substituted 1, Mono-substituted 2 and Di-substitutedobtained in Example 14 was digested with a protease by a proceduresimilar to the hG-CSF derivative peptide mapping described in Example 7.

As the results, disappearance or considerable reduction of specifiedpeaks was found when compared with the analytical pattern of hG-CSF aswas found in Example 8.

According to a method similar to that in Example 8, the binding positionof polyethylene glycol was estimated as follows.

Mono-substituted 1:

The position was within a peptide fragment (not including free aminogroups of lysine and the like) corresponding to the 47 to 93 positionresidues of the amino acid sequence represented by SEQ ID NO:1, and itwas confirmed according to the method described in Example 12 that thehydroxyl group of the 66 position Ser counting from the N-terminus wasmodified with one molecule of polyethylene glycol.

Mono-substituted 2:

The position was within a peptide fragment (including an N-terminalamino group) corresponding to the −1 to 19 position residues of theamino acid sequence represented by SEQ ID NO:1, and it was confirmedaccording to the method described in Example 7 that the N-terminal Metwas modified with one molecule of polyethylene glycol.

Di-substituted:

A peptide fragment (including an N-terminal amino group) correspondingto the −1 to 19 position residues, and a peptide fragment (not includingfree amino groups of lysine and the like residues) corresponding to the47 to 93 position residues, of the amino acid sequence represented bySEQ ID NO:1.

Example 16

Preparation of polyethylene glycol-modified hG-CSF derivative reactionsolution:

The hG-CSF derivative prepared in Reference Example 1 was adjusted to0.9 mg/ml using a phosphate buffer (pH 7.2) to obtain 600 ml of theadjusted solution. While cooling in an ice bath, the thus obtainedsolution was mixed with 8.7 g of N-hydroxysuccinimide ester of 2,4-bis(o-methoxy-polyethylene glycol)-6-(3-carboxypropylamino)-s-triazineobtained by a method similar to that in Reference Example 2, and themixture was stirred at 4° C. for 48 hours to prepare a polyethyleneglycol-modified hG-CSF derivative reaction solution (4).

Example 17

Isolation of polyethylene glycol-modified component:

The polyethylene glycol-modified hG-CSF derivative reaction solution (4)prepared by a method similar to that in Example 16 (protein content, 40mg) was passed through a column packed with Sephacryl S-300(manufactured by Pharmacia) which had been equilibrated in advance witha 20 mM acetate buffer (pH 4.5) containing 150 mM of sodium chloride,and the eluates were fractionated. By this operation, a crude fractioncontaining a component modified with three molecules of polyethyleneglycol (Tri-substituted) and a crude fraction containing a componentmodified with two molecules of polyethylene glycol (Di-substituted) wereobtained.

Each of these fractions was purified using a cation exchange columnSP-5PW (manufactured by Tosoh Corp.) in a manner similar to that inExample 4 to obtain two species of the main components ofTri-substituted (Tri-substituted 1: 0.49 mg/ml×1.3 ml, Tri-substituted2: 0.57 mg/ml×1.5 ml) and one species of the main component ofDi-substituted (1.94 mg/ml×1.2 ml).

Example 18

Estimation of polyethylene glycol binding position by peptide mapping:

Polyethylene glycol-binding positions of Di-substituted, Tri-substituted1 and Tri-substituted 2 obtained in Example 17 were examined by aprocedure similar to the peptide mapping described in Examples 7 and 8.

Also in Di-substituted, Tri-substituted 1 and Tri-substituted 2 obtainedin Example 17, it was estimated that polyethylene glycol was bound to apeptide fragment (including an N-terminal amino group) corresponding tothe −1 to 19 position residues of the hG-CSF derivative and to a peptidefragment (not including free amino groups of lysine and the like)corresponding to the 47 to 93 position residues of the hG-CSFderivative. In Tri-substituted 1 and Tri-substituted 2, it was estimatedthat polyethylene glycol was also bound to at least a peptide fragment(including an amino group of Lys) corresponding to the 34 to 46 positionresidues (peak 3) of the hG-CSF derivative.

Example 19

Preparation of polyethylene glycol-modified hG-CSF derivative reactionsolution:

The hG-CSF derivative prepared in Reference Example 1 was adjusted to0.9 mg/ml using a phosphate buffer (pH 7.3) to obtain 560 ml of theadjusted solution. While cooling in an ice bath, the thus obtainedsolution was mixed with 22.4 g of N-hydroxysuccinimide ester of2,4-bis(o-methoxypolyethyleneglycol)-6-(3-carboxypropyl-amino)-s-triazine obtained by a methoddescribed similar to that in Reference Example 4, and the mixture wasstirred at 4° C. for 48 hours to prepare a polyethylene glycol-modifiedhG-CSF derivative reaction solution (5).

Example 20

Isolation of polyethylene glycol-modified component:

The polyethylene glycol-modified hG-CSF derivative reaction solution (5)prepared by a method similar to that in Example 19 (protein content, 15mg) was passed through a column packed with Sephacryl S-400(manufactured by Pharmacia) which had been equilibrated in advance witha 20 mM acetate buffer (pH 4.5) containing 150 mM of sodium chloride,and the eluates were fractionated. By this operation, a crude fractioncontaining a component modified with three molecules of polyethyleneglycol (Tri-substituted) and a crude fraction containing a componentmodified with two molecules of polyethylene glycol (Di-substituted) wereobtained.

Each of these fractions was purified using a cation exchange columnSP-5PW (manufactured by Tosoh Corp.) in a manner similar to that inExample 4 to obtain two species of the main components ofTri-substituted (Tri-substituted 1:3.53 mg/ml×0.4 ml, Tri-substituted 2:0.26 mg/ml×2.1 ml) and one species of the main component ofDi-substituted (0.84 mg/ml×1.2 ml).

Example 21

Estimation of polyethylene glycol binding position by peptide mapping:

Polyethylene glycol-binding positions of Di-substituted, Tri-substituted1 and Tri-substituted 2 obtained in Example 20 were examined by aprocedure similar to the peptide mapping described in Examples 7 and 8.Also in Di-substituted, Tri-substituted 1 and Tri-substituted 2 obtainedin Example 20, it was estimated that polyethylene glycol was bound to apeptide fragment (including an N-terminal amino group) corresponding tothe −1 to 19 position residues of the hG-CSF derivative and to a peptidefragment (not including free amino groups of lysine and the like)corresponding to the 47 to 93 position residues of the hG-CSFderivative. In Tri-substituted 1 and Tri-substituted 2, it was estimatedthat polyethylene glycol was also bound to at least a peptide fragment(including an amino group of Lys) corresponding to the 34 to 46 positionresidues of the hG-CSF derivative.

Example 22

Protease resistance of chemically modified polypeptide:

A 0.5 ml portion of each of the two species of Mono-substitutedcomponents obtained in Example 14 was passed through a gel filtrationcolumn [Sephadex G-25 (NAP-5, manufactured by Amersham-Pharmacia)] whichhad been equilibrated with a 10 mM phosphate buffer (pH 5.0), and theeluates were recovered in 1.0 ml portions.

Each of the thus recovered fractions was diluted with a 10 mM phosphatebuffer (pH 5.0) to adjust the protein concentration to 0.2 mg/ml.

A 2 ml portion of each of the thus diluted fractions was mixed with 0.2mg/ml of thermolysin (enzyme/substrate ratio=1/50) and the mixture wasallowed to react at 30° C.

Samples were periodically collected from the reaction solutions in 100μl portions and mixed with 2 μl of acetic acid to terminate thereaction.

Using 50 μl of each of the reaction-terminated samples, HPLC analysiswas carried out under the following conditions.

HPLC analysis conditions:

Column: TSK gel G4000SW_(XL) (7.8 mm I.D.×300 mm) (manufactured byTosoh)

Detection: 280 nm

Mobile phase: 150 mM NaCl/20 mM sodium acetate buffer (pH 4.5)

Flow rate: 0.8 ml/min

Mono-substituted's 1 and 2 were eluted at a retention time of 12minutes. When these Mono-substituted's were hydrolyzed by thermolysin,the detection peak found at the position of 12 minutes decreases so thatthe degree of hydrolysis of these Mono substituted's by thermolysincould be confirmed.

Table 9 shows periodic changes in the residual ratio ofMono-substituted's calculated from the decreasing ratio of the detectionpeak found at the position of 12 minutes.

TABLE 9 Stability against thermolysin Reaction time Mono-substituted 1Mono-substituted 2 (hr) of Ex. 14 of Ex. 14 0 100 100 15 95 72 23 89 68

It was found from the results shown in Table 9 that Mono-substituted 1in which Ser was modified with polyethylene glycol was more resistant tothermolysin than Mono-substituted 2 in which the N-terminus wasmodified.

Example 23

Stability of chemically modified polypeptide against freezing-thawing:

A 1 ml portion of each of the two species of Mono-substituted componentsobtained in Example 5 was passed through two gel filtration columns[Sephadex G-25 (NAP-10, manufactured by Amersham-Pharmacia)] which hadbeen equilibrated with a 5 mM sodium acetate buffer (pH 4.5) or a 5 mMsodium acetate buffer (pH 5.0), and the eluates from each column wererecovered in 1.5 ml portions.

The protein concentration of each of the thus recovered fractions wasadjusted to 300 μg/ml.

Each of the thus adjusted fractions was dispensed in 500 μl portions,frozen at −30° C. and then melted in a water bath at room temperature.

This freezing-thawing treatment was repeated four times, and then theremaining amount (recovery yield) of each modified polypeptide wasmeasured under the gel filtration HPLC conditions described in Example22.

The results are shown in Table 10.

TABLE 10 Polyethylene Recovery yield (%) after four glycol-modifiedfreezing-melting treatments product pH 4.5 pH 5.0 Mono-substituted 1 100101 of Ex. 5 Mono-substituted 2 91.3 85.5 of Ex. 5

It was found from the results shown in Table 10 that Mono-substituted 1in which Ser was modified with polyethylene glycol was more stableagainst freezing-thawing than Mono-substituted 2 in which the N-terminuswas modified.

Example 24

In vitro activity of chemically modified polypeptide:

The activity of the two species of Mono-substituted components obtainedin Example 14 to enhance growth of mouse leukemia cell NFS60 [K. Holmeset al., Proc. Natl. Acad. Sci. USA, 82:6687 (1985)] was measured inaccordance with the method of Asano et al. [Yakuri to Chiryo, 19:2767(1991)] by the following serial dilution method.

A suspension of the cells washed with G-CSF (−) medium was dispensed in50 μl portions into wells of a 96 well plate.

Mono-substituted 1 obtained in Example 14 was adjusted to 25 ng/ml, anda 50 μl portion of the adjusted solution was added to the first well andthoroughly mixed to adjust the solution to 12.5 ng/ml.

A 50 μl portion of the solution was removed from the well and added tothe second well and thoroughly mixed to adjust the solution to 6.25ng/ml. By repeating this procedure, 11 steps of two-fold dilution serieswere prepared.

In the same manner, two-fold dilution series were prepared usingMono-substituted 2 (25 ng/ml) obtained in Example 14 and a standardsolution (5 ng/ml) containing the hG-CSF derivative of ReferenceExample 1. By this method, dilution series of Mono-substituted 2 from12.5 ng/ml and dilution series of the standard solution from 2.5 ng/mlwere prepared in 50 μl portions in respective wells.

The growth activity of NFS60 cells was measured three times for each ofthe sample solutions and the standard solutions, and relative activityof each Mono-substituted was calculated based on the activity (=100) ofthe standard solution.

Mono-substituted 1 showed 1.06 to 1.13 times higher activity thanMono-substituted 2.

Reference Example 1

In accordance with the method described in Reference Example 3 ofJapanese Examined Patent Application No. 096558/95, a hG-CSF derivative(compound k in Table 1) was obtained from hG-CSF having the amino acidsequence shown in SEQ ID NO:1 by replacing the 1 position threonine withalanine, the 3 position leucine with threonine, the 4 position glycinewith tyrosine, the 5 position proline with arginine and the 17 positioncystine with serine.

That is, a strain of Escherichia coli, W3110 strA (Escherichia coliECfBD28, FERM BP-1479), which has a plasmid pCfBD28 containing a DNAfragment coding for the above-described hG-CSF derivative was culturedat 37° C. for 18 hours in LG medium [prepared by dissolving 10 g ofBacto Tryptone, 5 g of Yeast Extract, 5 g of sodium chloride and 1 g ofglucose in 1 liter of water and adjusting its pH to 7.9 with NaOH], a 5ml portion of the cultured broth was inoculated into 100 ml of MCGmedium (0.6% Na₂HPO₄, 0.3% KH₂PO₄, 0.5% sodium chloride, 0.5% casaminoacid, 1 mM MgSO₄ and 4 μg/ml of vitamin B1, pH 7.2) containing 25 μg/mlof tryptophan and 50 μg/ml of ampicillin and the mixture was cultured at30° C. for 4 to 8 hours. Then, 10 μg/ml of a tryptophan inducer3β-indoleacrylic acid (hereinafter referred to as “IAA”) was added tothe medium and the culturing was continued for additional 2 to 12 hours.The resulting culture broth was centrifuged at 8,000 rpm for 10 minutes,and the thus collected cells were washed with 30 mM sodium chloride anda 30 mM Tris-HCl buffer (pH 7.5). The thus washed cells were suspendedin 30 ml of the above-described buffer and disrupted ultrasonically at0° C. for 10 minutes using a sonicator (SINIFIER CELL DISRUPTOR 200,OUTPUT CONTROL 2, manufactured by BRANSON SONIC POWER COMPANY). The cellsuspension after the ultrasonic disruption was centrifuged at 9,000 rpmfor 30 minutes to obtain the cell residue.

Thereafter, the hG-CSF derivative was extracted and purified from thecell residue and solubilized and regenerated, in accordance with themethod of Marston et al. [F. A. O. Marston et al., BIO/TECHNOLOGY, 2:800(1984)].

Reference Example 2

Production of N-hydroxysuccinimide ester of2,4-bis(o-methoxypolyethyleneglycol)-6-(3-carboxy-propylamino)-s-triazine:

412 mg (4.0 mmol) of γ-aminobutyric acid was dissolved in 300 ml of a0.1 M borate buffer (pH 10), and the resulting solution which was cooledin an ice bath was mixed with 20 g (2 mmol) of2,4-bis(o-methoxypolyethylene glycol)-6-chloro-s-triazine (manufacturedby Seikagaku Kogyo) and the mixture was allowed to react at 4° C. for awhole day and night and then at room temperature for 6 hours.

The reaction solution was adjusted to pH 1 by adding 1 N hydrochloricacid, and then 2,4-bis(o-methoxypolyethyleneglycol)-6-(3-carboxypropyl-amino)-s-triazine was extracted withchloroform. The chloroform phase was dried over anhydrous sodiumsulfate, the insoluble matters were removed by filtration, and then thesolvent was evaporated under reduced pressure to obtain the carboxylicacid derivative.

The carboxylic acid derivative was recrystallized from dry acetone toobtain 15.8 g (1.6 mmol) of crystals of the carboxylic acid derivative.

A 10 g (1.0 mmol) portion of the crystals and 230 mg ofN-hydroxysuccinimide were dissolved in 100 ml of anhydrous methylenechloride and, while cooling in an ice bath and in a stream of argon, theresulting solution was mixed with 413 mg ofN,N′-dicyclohexylcarbodiimide (DCC) and the mixture was stirred for 30minutes. After completion of the stirring, the temperature of thereaction mixture was returned to room temperature, and stirred foradditional 1.5 hours, the insoluble matters (N,N′-dicyclohexylurea(DCU)) were removed by filtration and then the resulting filtrate wasconcentrated to 40 ml under reduced pressure.

The concentrate was added dropwise to 600 ml of anhydrous diethyl etherto form a precipitate. The precipitate was washed with anhydrous diethylether and then the solvent was removed under reduced pressure to obtain7.7 g (0.77 mmol) of N-hydroxysuccinimide ester of2,4-bis(o-methoxy-polyethyleneglycol)-6-(3-carboxypropylamino)-s-triazine (yield, 77%).

Reference Example 3

Production of 2,4-bis(o-methoxypolyethyleneglycol)-6-(3-carboxypropylamino)-s-triazine:

100 g (8.33 mmol) of thoroughly dried monomethoxypolyethylene glycolhaving an average molecular weight of 12,000 (manufactured by Nippon Oil& Fats), 9.3 g of zinc oxide (Wako Pure Chemical Industries) and 83.5 gof molecular sieves (Type 4A) (Wako Pure Chemical Industries) weredissolved in dry benzene, and the solution was allowed to stand at roomtemperature for a whole day and night in a stream of argon.

After removing the molecular sieves, the reaction solution was distilledat 80° C. in a stream of argon using a distillation apparatus and thenazeotropically distilled for a whole day and night at 80° C. in a streamof argon using a Soxhlet extractor (for solid phase use) which had beenpacked with about 100 g of molecular sieves (Type 4A).

The reaction solution obtained by the dehydration reflux was cooled,mixed with 736 mg (4.0 mmol) of cyanuric chloride and thenazeotropically distilled for 5 days in a manner similar to thatdescribed above. Thereafter, this solution was cooled to roomtemperature, mixed with 300 ml of dry benzene and then the mixture wascentrifuged at 3,600 rpm for 10 minutes to remove insoluble matter.

The thus obtained supernatant was concentrated to 300 ml under reducedpressure and added dropwise to 3,000 ml of dry diethyl ether to form aprecipitate. The precipitate was recovered and washed with dry diethylether, and then the solvent was removed under reduced pressure to obtaina dry precipitate.

While cooling in an ice bath, 100 g of the dry precipitate was added toa solution prepared by dissolving 1.24 g (12.0 mmol) of γ-aminobutyricacid in 1,000 ml of a 0.1 M borate buffer (pH 10), and the mixture wasstirred at 4° C. for a whole day and night. After additional 6 hours ofstirring at room temperature, this solution was adjusted to pH 1.0 with1 N hydrochloric acid and then 2,4-bis(o-methoxypolyethyleneglycol)-6-(3-carboxypropyl-amino)-s-triazine was extracted withchloroform.

The chloroform phase containing the compound was dried over anhydroussodium sulfate and filtered, and then the solvent was removed from theresulting filtrate under reduced pressure. The thus formed white solidwas recrystallized from dry acetone to obtain about 90 g of2,4-bis(o-methoxy-polyethyleneglycol)-6-(3-carboxypropylamino)-s-triazine (yield, 90%).

Reference Example 4

Production of N-hydroxysuccinimide ester of2,4-bis(o-methoxypolyethyleneglycol)-6-(3-carboxy-propylamino)-s-triazine:

25 g (1.0 mmol) of 2,4-bis(o-methoxy-polyethyleneglycol)-6-(3-carboxypropylamino)-s-triazine which had been synthesizedin a manner similar to that in Reference Example 3 and thoroughly driedand 240 mg of N-hydroxysuccinimide were dissolved in 400 ml of anhydrousmethylene chloride and, while cooling in an ice bath and in a stream ofargon, the resulting solution was mixed with 431 mg ofN,N′-dicyclohexylcarbodiimide (DCC) and the mixture was stirred for 30minutes.

After completion of the stirring, the reaction mixture was returned toroom temperature and stirred for additional 1.5 hours, the insolublematters (N,N′-dicyclohexylurea (DCU)) were removed by filtration andthen the resulting filtrate was concentrated to 160 ml under reducedpressure.

The concentrate was added dropwise to 2,400 ml of anhydrous diethylether to form a precipitate, the precipitate was washed with anhydrousdiethyl ether and then the solvent was removed under reduced pressure toobtain 21.4 g (0.89 mmol) of N-hydroxysuccinimide ester of2,4-bis(o-methoxypolyethyleneglycol)-6-(3-carboxy-propylamino)-s-triazine (yield, 89%).

INDUSTRIAL APPLICABILITY

The present invention provides a chemically modified polypeptide inwhich at least one of the hydroxyl groups in the polypeptide molecule ismodified with a polyalkylene glycol derivative; a method for producingthe modified polypeptide; a method for treatment using the modifiedpolypeptide; use of the modified polypeptide; a pharmaceuticalpreparation comprising the modified polypeptide; and a composition fortreatment comprising the modified polypeptide.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 1 <210> SEQ ID NO 1 <211> LENGTH: 175<212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: polypeptide      having hG-CSF activity <400> SEQUENCE: 1Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pr #o Gln Ser Phe Leu Leu- 1   1               # 5                  # 10                  # 15Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gl #y Asp Gly Ala Ala Leu                 20  #                 25  #                 30Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cy #s His Pro Glu Glu Leu             35      #             40      #             45Val Leu Leu Gly His Ser Leu Gly Ile Pro Tr #p Ala Pro Leu Ser Ser         50          #         55          #         60Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cy #s Leu Ser Gln Leu His     65              #     70              #     75Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gl #n Ala Leu Glu Gly Ile 80                  # 85                  # 90                  # 95Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Le #u Gln Leu Asp Val Ala                100   #               105   #               110Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Gl #u Glu Leu Gly Met Ala            115       #           120       #           125Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pr #o Ala Phe Ala Ser Ala        130           #       135           #       140Phe Gln Arg Arg Ala Gly Gly Val Leu Val Al #a Ser His Leu Gln Ser    145               #   150               #   155Phe Leu Glu Val Ser Tyr Arg Val Leu Arg Hi #s Leu Ala Gln Pro160                 1 #65                 1 #70

What is claimed is:
 1. A chemically modified polypeptide having agranulocyte colony-stimulated factor activity in which the hydroxylgroup of at least one serine residue is modified with a polyalkyleneglycol derivative.
 2. The chemically modified polypeptide according toclaim 1, wherein the polypeptide having a granulocyte colony-stimulatingfactor activity is a polypeptide comprising the amino acid sequencerepresented by SEQ ID NO: 1, or a polypeptide comprising an amino acidsequence in which at least one amino acid is deleted, substituted oradded in the amino acid sequence represented by SEQ ID NO: 1, and havinga granulocyte colony-stimulating factor activity.
 3. The chemicallymodified polypeptide according to claim 1, wherein the polyallcyleneglycol derivative has a molecular weight of 500 to 1,000,000.
 4. Thechemically modified polypeptide according to claim 1, which is modifiedwith a chemical modifying agent comprising a polyalkylene glycolderivative.
 5. The chemically modified polypeptide according to claim 4,wherein the chemical modifying agent is a polyalkylene glycol derivativerepresented by the following formula (I): R¹—(M)_(n)—X—R²  (I) whereinR1 represents an alkyl group or an alkanoyl group; M represents—OCH₂CH₂—, —OCH₂CH₂CH₂— or —(OCH₂CH₂)_(r)—(OCH₂CH₂CH₂)₈— wherein r and sare the same or different, and each represents an optionally changeablepositive integer; n is an optionally changeable positive integer; Xrepresents a bond, O, NH or S; and R² represents

wherein R³ represents OH, halogen or —X^(a)—(M^(a))_(na)—R^(1a) whereinX^(a), M^(a), R^(1a) and na each have the same meanings as the above X,M, R¹ 1 and n, respectively; and Y represents halogen or—Z—(CH₂)_(p)—(O)_(m)—W wherein Z represents O, S or NH; and W representsa carboxyl group or a reactive derivative thereof, or

wherein R⁴ represents an alkyl group, and Hal represents halogen; p isan integer of 0 to 6; and m is 0 or 1, —(CO)_(m)—(CH₂)_(t)—W wherein tis an integer of 0 to 6

wherein Hal^(a), pa and R^(4a) each has the same meanings as the aboveHal, p and R⁴, respectively

wherein R⁵ represents a residue in which an amino group and a carboxylgroup are removed from an amino acid.
 6. The chemically modifiedpolypeptide according to claim 4, wherein the chemical modifying agentis a polyalkylene glycol derivative represented by the following formula(Ib):

wherein W^(a) represents a carboxyl group or a reactive derivativethereof.
 7. A pharmaceutical composition comprising a chemicallymodified polypeptide according to claim 1 together with apharmaceutically acceptable carrier.
 8. A method of improving resistanceof a polypeptide having a granulocyte colony-stimulating factor activityto protease, freezing-thawing or denaturating agents, said methodcomprising modifying the hydroxyl group of at least one serine residuewith a polyalicylene glycol derivative.